Microorganisms isolated for the purpose of clinical diagnostics, as well as those isolated to monitor contamination of foodstuffs, medical tissues, or the environment, often need to be characterized in order to determine the appropriate response to the presence of the organisms found. Traditional automated phenotypic identification assays, such as the Vitek®, Phoenix™, and Microscan® systems, or manual phenotypic tests such as API, require that microorganisms be in an appropriate growth phase and free of interfering media and blood products in order to provide robust results. These systems use colonies grown for 16-24 hours on plated media, after which standardized suspensions are made from the colonies, and then the actual characterization tests require a further 4-24 hours of incubation to complete.
Optical spectroscopy methods, such as intrinsic fluorescence (IF), infrared spectroscopy (FTIR), or Raman spectroscopy, have the potential to allow for identification of microorganisms very quickly, but have only been demonstrated to work with “clean” microorganism suspensions. Publications have described IF methods for microbial characterization with only very limited organism sets, or that required additional measures, such as specific binding events, to allow functional characterization. Direct examination of microorganisms on growth medium has been considered problematic due to the assumed large contribution of the medium itself to the spectroscopic pattern.
The present invention overcomes the problems in the art by providing methods that can discriminate between microorganisms spectroscopically interrogated directly on fluorescent solid and/or semi-solid growth media, including highly fluorescent media.